This invention relates to a process for producing a natural blue pigment by submerged fermentation of a mutant Morel mushroom fungus, Morchella nov. ES-1 sp. This vivid blue pigment is useful in food, drug, medical device, cosmetic and textile coloring or dying applications. The pigment has been identified as indigo (or indigotin, as it is also called).
The use of synthetic pigments in cosmetic, food and medical applications is increasingly questioned for safety reasons. Various synthetic coloring agents have been banned as edible coloring agents due to potential carcinogenicity or teratogenicity. In addition, publicity and consumer education on this topic has led to increasing interest in and demands for the use of safer, naturally occurring pigments and coloring agents.
Indigo is a naturally occurring glucoside found in many plants, particularly of the genera Indigofera and Isatus. Indigo released from such plants has been used as a textile dye for thousands of years.
Naturally occurring pigments typically have certain limitations, however. They may be of lower tinctorial strength than synthetic colorants. In addition, the pH of various food and cosmetic products is known to affect the shade of the pigment, as well as its stability to heat, light and preservatives.
The production of indigo by submerged culture of bacterial strains (e.g., Pseudomonas indoloxidans, Mycobacterium globerulum, Micrococcus oiltonensis) isolated from soil samples using indole as a source of carbon and nitrogen in the medium has been reported by Gray, "Formation of Indigotin from Indole by Soil Bacteria," Roy. Soc. Proc., 102:263-80(1972).
U.S. Pat. No. 4,520,103 (Ensley, Jr.) reported microbiological production of indigo in a genetically-transformed microorganism grown in an indole-free medium. The microorganism used was selected on the basis of having the metabolic capacity to produce and accumulate indole. The genetic transformation incorporated the capacity to synthesize one or more aromatic dioxygenase enzymes which catalyze the oxidative transformation of the accumulated indole. The reaction product is further processed by the cell to indigo.
Miles et al., "The Identification of Indigo as a Pigment Produced by a Mutant Culture of Schizophyllum commune," Arch. Biochem. and Biophys., 62:1-5(1956), identified as indigo a blue pigment harvested from mycelial macerates of a mutant culture of Schizophyllum commune fungus. Falanghe et al., "Identification of Indigo Produced in Submerged Culture of Agaricus campestris, Mutant Culture," Arch. of Biochem. and Biophys., 96:430-33(1962), isolated a single mutant strain of Agaricus camoestris which produced a blue color in the medium. The blue coloring matter was isolated and separated into three main fractions, one of which was identified as indigo.